What is automated nucleic acid purification system?
are one of the most basic substances of life and can be divided into two categories: DNA and RNA. Nucleic acids are widely found in all living things such as animal and plant cells and microorganisms. It not only plays the role of storing and transmitting genetic information but also occupies an important position in protein biosynthesis, thus playing a decisive role in a series of major life phenomena such as growth, heredity, and mutation. Nucleic acid extraction
includes DNA extraction, RNA extraction, and plasmid extraction. Nucleic acids are the carriers of genetic information, the material basis of gene expression, and the main object of molecular biology research. Whether conducting structural or functional studies of nucleic acids, they need to be extracted and purified first. Nucleic acid purification systems
are instruments that automatically complete the extraction of nucleic acids from samples by applying a matching nucleic acid extraction reagent. There are two types of nucleic acid extractors: large automated ones, generally called automated liquid workstations and small automated nucleic acid purification systems
that use encapsulated reagents to automate the extraction and purification process. Largely automated liquid workstations are used less and less because of the high cost of equipment, high operating costs, and the suitability of extracting thousands of specimens of the same species at a time, while small automated instruments are increasingly used because of the low cost of equipment and operation, and ease of operation.
Nucleic acid purification system
Nucleic acid extraction and purification method
Since the first discovery of nucleic acids in 1869, many researchers have made unremitting efforts to explore the extraction methods of nucleic acids and improved various materials and reagents for nucleic acids. Various reagents such as sodium dodecyl sulfate, phenol, urea, and guanidine salts have been applied to nucleic acid extraction experiments, and various commercial kits for nucleic acid extraction have been developed. Among them, the traditional extraction methods mainly include phenol extraction, alkali lysis, CTAB extraction, and EtBr-CsCl gradient centrifugation. These traditional extraction methods can isolate DNA and RNA from different tissue samples, but these techniques include precipitation and centrifugation, which require a large volume of biological samples, and the extraction steps are tedious, time-consuming, and laborious, and the yield is not high, making it difficult to automate the operation. In addition, most of the traditional methods require the use of toxic chemical reagents, which are potentially harmful to the health of operators. Therefore, along with the development of molecular biology and polymeric materials, the traditional techniques for the separation of nucleic acids from liquid-phase systems are gradually being replaced by new methods based on solid-phase carriers.
Solid-phase carrier adsorption method
The new nucleic acid extraction methods based on solid-phase sorbent carriers include rotary centrifugal column extraction, glass bead adsorption, silica matrix method, anion exchange method, and magnetic nano-bead extraction method. The operation steps of these methods can be divided into three main parts.
a. Use lysis solution to induce cell rupture to release nucleic acids in the liquid phase.
b. Using the strong affinity and adsorption of the carrier for nucleic acids, the released nucleic acids are specifically bound to the specific carrier, so that other impurities such as proteins, polysaccharides, and lipids remain free in the liquid phase and are removed with the removal of the supernatant.
c. The purified nucleic acids are obtained by adjusting the ionic strength and pH of the eluent to elute the nucleic acids adsorbed on the carrier.
Among them, centrifugal column method DNA extraction kits are widely used in the market due to their low price and relatively convenient operation. However, as the demand for DNA extraction increases, the disadvantages of the centrifugal column method for DNA extraction are becoming more and more prominent. The large volume of samples required, the loss of many samples and the inability to work with rare samples have become unavoidable disadvantages of the centrifugal column method, while the process of DNA extraction by centrifugal column method requires repeated centrifugation, which is not convenient for high-throughput and automated operation and is not compatible with the requirements of modern biology experiments.
Advantages of magnetic bead method for nucleic acid extraction
Magnetic bead method of DNA extraction is a perfect combination of nanotechnology and biotechnology, with unparalleled advantages over other DNA extraction methods.
a. Low sample requirement
High concentrations of nucleic acids can be mentioned with a minute amount of material.
b. Simple and fast operation
The whole operation process is divided into five steps (lysis, binding, washing, drying, and elution), and the whole process can be completed within 30~60 minutes without centrifugal operation mostly.
c. Stable and reliable quality
The free magnetic beads bind more to nucleic acid, the specific binding makes nucleic acid purity higher, and the amount of nucleic acid recovery can be adjusted by controlling the surface groups of magnetic beads.
d. Fully automated operation
The use of a nucleic acid extractor can realize automatic, high-throughput operation, a key start, you can realize the extraction of dozens or even hundreds of samples.
e. Safe, non-toxic and harmless
The reagent does not contain phenol, chloroform, and other toxic chemical reagents, which is in full compliance with modern environmental protection concepts.
Notes on nucleic acid extraction by magnetic bead method
Error 1: The more magnetic beads are used, the better the purification effect
The main characteristic of magnetic beads is that they can be dispersed in the liquid, and also can be separated from the liquid phase in a solid state under the action of the applied magnetic field. In any reagent system, the ratio of magnetic beads to liquid should be a certain threshold, beyond a certain ratio, too many magnetic beads will lose their dispersion characteristics because they cannot be evenly dispersed in the liquid, which also makes the washing process unable to fully increase the efficiency of the nucleic acid magnetic beads and liquid contact. Excessive magnetic beads will also adsorb more impurities, which will have a great impact on the effect of debridement. There are even times when too many magnetic beads will adsorb functional components that play a major role in the liquid system such as protease and lysozyme, resulting in the inefficiency of the whole kit. There are many times, when the purification is not effective, reducing the number of magnetic beads used is instead the optimal way to improve the purification effect.
Usually, the reference bead dosage given by the magnetic bead method kit is slightly excessive, so there are not many cases when it is necessary to increase the bead dosage to improve the adsorption efficiency, but if it is determined that the poor purification is caused by insufficient bead dosage, it is possible to improve the purification by increasing the bead dosage within a certain range.
Error 2: The more reagents used, the better the purification effect
For the magnetic bead method, every increase in a portion of the liquid volume reduces the chance of more bead collisions and reducing the chance of bead collisions leads to a substantial decrease in the adsorption rate. Therefore, in many cases, although increasing the lysis solution and washing solution can indeed play a role in enhancing lysis and enhancing washing, the core of the magnetic bead method purification is the efficiency of nucleic acid adsorption by magnetic beads, and the inability to ensure the efficiency of magnetic bead collision cannot guarantee the efficiency of nucleic acid purification, so simply increasing the number of reagents used to improve the purification effect is not necessarily fully effective.
Error 3: The more washes, the better the purification effect
Increasing the number of washes is indeed beneficial to the purification of nucleic acids, but considering that each wash loses a certain number of nucleic acids and increases the possibility of nucleic acid breakage and hydrolysis, it is generally appropriate to control the number of washes to 2~4 times.
Error 4: The more samples are taken, the better the purification effect
When the sample is not fresh enough or the nucleic acid content itself is very small, more samples are often taken to increase the nucleic acid extraction volume when the nucleic acid extraction is not effective. However, simply increasing the sample sampling volume can sometimes introduce too many impurities beyond the lysis capacity of the lysate and also reduce the extraction efficiency, so it is not recommended to increase the extraction volume by simply increasing the sample sampling volume. If the extraction volume is indeed too low due to insufficient sample size, it is recommended to go through an enrichment or concentration step before starting the extraction in the pretreatment. Alternatively, increasing the completeness of the lysis so that more nucleic acids are exposed is also a solution.
Core parameters of nucleic acid purification system
a. Extraction principle
b. Mechanical principle
c. Extraction time
d. Sample throughput
e. Sample tube capacity
f. Tip head capacity
g. Number of Tip Heads
i. Instrument weight
j. Instrument size
Application areas of nucleic acid extraction and purification
In almost every laboratory, the separation and purification work related to biomolecules is very important and essential. However, it is still quite difficult to purify multiple samples, which not only requires the selection of appropriate purification techniques but also an exceptionally high workload, making it difficult to meet the current rapidly developing demand for extraction and purification of high-throughput samples.
The automated nucleic acid purification system is ideal for genomics research. Samples can be extracted from a microbial, animal, plant, or viral sources. Whole blood genomic DNA extraction kits, leukocyte layer whole blood genomic extraction kits, and animal tissue/cell genomic DNA extraction kits can be used to quickly purify DNA or RNA in sufficient quantity and purity.
The automated nucleic acid purification system can be used to address rapid automated epidemic surveillance systems for Influenza A virus subtype H1N1, childhood hand, foot, and mouth disease, and measles viruses to improve response capabilities for major epidemics.
c. Molecular diagnosis of clinical samples
The automated nucleic acid purification system can process clinical samples quickly and with high throughput, and the extracted nucleic acids can be used for subsequent molecular diagnosis.
d. Animal husbandry and veterinary medicine
The automated nucleic acid purification system can extract avian influenza virus, Newcastle disease virus, classical swine fever virus (CSFV), bovine viral diarrhea virus, rickettsiae, etc. with high efficiency and sensitivity. viral diarrhea virus), rickettsiae (Coxiella burnetii), etc.
e. Applications in forensic medicine
The efficiency and stability of nucleic acid extraction is very important for forensic work. The automated nucleic acid purification system works in conjunction with specialized magnetic bead reagents for nucleic acid extraction from forensic samples to purify high-quality DNA from materials of different origins including cigarette butts, hair roots, cartilage, fingernails, blood traces, etc.
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